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. 2019 Nov 1;17(11):e3000499. doi: 10.1371/journal.pbio.3000499

Fig 3. Bimolecular fluorescence shows in vivo interaction between UNC-62 and CEH-60 in the adult intestine.

Fig 3

(A) Bright-field and YFP images of animals carrying hsp-16.41p::ceh-20::VC155 and hsp-16.41p::unc-62::VN173. In vivo interaction between CEH-20 and UNC-62 reconstitutes fluorescence ubiquitously after heat shock (33°C, 2 hours), most clearly visible in the intestinal nuclei. (B) Interaction between CEH-60 and UNC-62 reconstitutes fluorescence in animals carrying hsp-16.41p::ceh-60::VC55 and hsp-16.41p::unc-62::VN173 after heat shock (33°C, 2 hours). (C) Interaction between CEH-60 and UNC-62 is not observed upon expression of a version of CEH-60 in which the PBC domain is not present, CEH-60(ΔPBC), indicating that this domain is needed for the interaction. (D) Quantification of Venus fluorescence for interaction between UNC-62 and CEH-20, CEH-60, or CEH-60(ΔPBC). One dot represents average fluorescence intensity in 6 intestinal nuclei per animal. ****p < 0.0001. N ≥ 6. Underlying data are available in S1 Data. (E) Bright-field and YFP images of animals carrying ceh-60p::ceh-60::VC155 and unc-62p::unc-62::VN173 transgenes, providing spatiotemporal specificity to the endogenous in vivo interaction of CEH-60 and UNC-62 in the adult intestine (solid arrows). No YFP signal was observed in other tissues besides the intestine. Reflecting the extrachromosomal nature of the transgenes, not all transgenic adult animals showed clear YFP in the intestine. Higher magnifications were used for endogenous BiFC because the YFP signal is much weaker compared to that of induced heat-shock promoters (A-C). Dotted arrow = signal of co-injection marker unc-122p::DsRed, * = fluorescent gut granule. Scale bar for A, B, and C = 100 μm. Scale bar for D = 50 μm. (F) Western blots detecting HA-tagged CEH-20, HA-tagged CEH-60, HA-tagged CEH-60(ΔPBC), or MYC-tagged UNC-62 in anti-HA immunoprecipitations using anti-HA and anti-MYC antibodies. CEH-20 and CEH-60, but not CEH-60(ΔPBC), co-immunoprecipitate UNC-62. A.U., arbitrary unit; BiFC, bimolecular fluorescence complementation; NS, not significant; YFP, yellow fluorescent protein.